The structural elements of the cell are never randomly distributed. Both the cytoplasm and nucleus are organized into different functional regions. The mechanisms of regionalization are important for understanding normal cellular physiology as well as disease states such as cancer or neurological diseases. Viruses take advantage of the regional organization within their host cells, resulting in enhanced virus replication and pathogenicity. The question of regionalization of the cytoplasm is a particularly important one for the nucleoprotein core (nucleocapsid) of negative strand RNA viruses because of the diversity of roles nucleocapsids play in the virus replication cycle. In particular, their roles as templates for viral RNA synthesis occur in regions of the cytoplasm that are distinct from the sites at which they are incorporated into progeny virions by budding from the host plasma membrane. Nucleocapsids are too large to diffuse freely in the cytoplasm, so there must be specific transport mechanisms to ensure their proper distribution. The proposed experiments address these mechanisms for the prototype negative strand RNA virus, vesicular stomatitis virus. We have developed two new cellular imaging approaches to quantify the movement of cellular elements to address these hypotheses. The first approach, which we call the border-to-border distribution method, quantifies the steady state distribution. The second involves improvement of live cell imaging approaches to determine the kinetics of particle movement that are particularly well suited to analyze actin-dependent motion. Using these approaches, our data show that nucleocapsids are transported toward the cell periphery by both actin filaments and microtubules. However, our data indicate that actin filaments are more important than microtubules in reaching the ultimate sites of virus assembly. Furthermore, the distribution of nucleocapsids in the cytoplasm appears to be coupled to membranes of the secretory pathway. These new analytical tools will be used in Aim 1 to determine the role of different myosin motors in establishing the distribution of nucleocapsids in the cytoplasm and incorporation into virions. Specific Aim 2 is to determine the dependence of nucleocapsid distribution on cellular membranes of the secretory pathway. These experiments will focus on GTP-binding proteins involved in membrane sorting and transport, and host membrane proteins associated with membrane-bound nucleocapsids identified by a series of new proteomics experiments. In Aim 3 the mechanisms of nucleocapsid distribution in polarized epithelial cells and neurons will be determined, since these represent the cell types involved in the natural infection by VSV. The proposed experiments challenge and seek to shift the current thinking on the mechanisms of interaction of viral capsids with the host cytoskeleton and membranes. They are also based on novel concepts and analytical methods that should be of general applicability in cell biology.